GluR4 Colorimetric Cell-Based ELISA Kit
- SKU:
- CBCAB00306
- Product Type:
- ELISA Kit
- ELISA Type:
- Cell Based
- Research Area:
- Signal Transduction
- Reactivity:
- Human
- Mouse
- Rat
- Detection Method:
- Colorimetric
Description
GluR4 Colorimetric Cell-Based ELISA Kit
The GLUR4 Colorimetric Cell-Based ELISA Kit is a cutting-edge tool designed for the precise measurement of GLUR4 levels in cell samples. This innovative kit offers exceptional sensitivity and specificity, guaranteeing accurate and consistent results for a variety of research applications.GLUR4, also known as glutamate receptor 4, is a key protein involved in synaptic transmission and neuronal plasticity. Dysregulation of GLUR4 has been implicated in various neurological disorders, such as epilepsy and Alzheimer's disease, highlighting its importance as a potential therapeutic target.
With the GLUR4 Colorimetric Cell-Based ELISA Kit, researchers can confidently study the role of GLUR4 in cellular processes and disease mechanisms, paving the way for new insights and therapeutic strategies in the field of neuroscience.
| Product Name: | GluR4 Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB00306 |
| ELISA Type: | Cell-Based |
| Target: | GluR4 |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The GluR4 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect GluR4 protein expression profile in cells. The kit can be used for measuring the relative amounts of GluR4 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on GluR4.
Qualitative determination of GluR4 concentration is achieved by an indirect ELISA format. In essence, GluR4 is captured by GluR4-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:
| 1. | A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values. |
| 2. | Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted. |
| Database Information: | Gene ID: 2893, UniProt ID: P48058, OMIM: 138246, Unigene: Hs.503743 |
| Gene Symbol: | GRIA4 |
| Sub Type: | None |
| UniProt Protein Function: | GluR4: an integral membrane protein belonging to the glutamate-gated ion channel family. L-glutamate (Glu) acts as an excitatory neurotransmitter at many synapses in the central nervous system. Glutamate receptors are heteromeric protein complexes with multiple subunits, each possessing transmembrane regions, and all arranged to form a ligand-gated ion channel. The postsynaptic actions of Glu are mediated by a variety of receptors that are named according to their selective agonists. This receptor binds AMPA(quisqualate) > glutamate > kainate. Each of the four GluR proteins (GRIA1-4) include flip and flop isoforms generated by alternative RNA splicing. |
| UniProt Protein Details: | Protein type:Membrane protein, integral; Channel, ligand-gated; Membrane protein, multi-pass Chromosomal Location of Human Ortholog: 11q22 Cellular Component: cell junction; cell soma; dendrite; plasma membrane; postsynaptic membrane; terminal button Molecular Function:alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate selective glutamate receptor activity; extracellular-glutamate-gated ion channel activity; ionotropic glutamate receptor activity Biological Process: glutamate signaling pathway; ionotropic glutamate receptor signaling pathway; synaptic transmission; transport |
| NCBI Summary: | Glutamate receptors are the predominant excitatory neurotransmitter receptors in the mammalian brain and are activated in a variety of normal neurophysiologic processes. These receptors are heteromeric protein complexes composed of multiple subunits, arranged to form ligand-gated ion channels. The classification of glutamate receptors is based on their activation by different pharmacologic agonists. The subunit encoded by this gene belongs to a family of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate)-sensitive glutamate receptors, and is subject to RNA editing (AGA->GGA; R->G). Alternative splicing of this gene results in transcript variants encoding different isoforms, which may vary in their signal transduction properties. Some haplotypes of this gene show a positive association with schizophrenia. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P48058 |
| NCBI GenInfo Identifier: | 218512059 |
| NCBI Gene ID: | 2893 |
| NCBI Accession: | P48058.2 |
| UniProt Secondary Accession: | P48058,Q86XE8, |
| UniProt Related Accession: | P48058 |
| Molecular Weight: | 49,146 Da |
| NCBI Full Name: | Glutamate receptor 4 |
| NCBI Synonym Full Names: | glutamate ionotropic receptor AMPA type subunit 4 |
| NCBI Official Symbol: | GRIA4 |
| NCBI Official Synonym Symbols: | GLUR4; GLURD; GluA4; GLUR4C |
| NCBI Protein Information: | glutamate receptor 4 |
| UniProt Protein Name: | Glutamate receptor 4 |
| UniProt Synonym Protein Names: | AMPA-selective glutamate receptor 4; GluR-D; Glutamate receptor ionotropic, AMPA 4; GluA4 |
| Protein Family: | Glutamate receptor |
| UniProt Gene Name: | GRIA4 |
| UniProt Entry Name: | GRIA4_HUMAN |
| Component | Quantity |
| 96-Well Cell Culture Clear-Bottom Microplate | 2 plates |
| 10X TBS | 24 mL |
| Quenching Buffer | 24 mL |
| Blocking Buffer | 50 mL |
| 15X Wash Buffer | 50 mL |
| Primary Antibody Diluent | 12 mL |
| 100x Anti-Phospho Target Antibody | 60 µL |
| 100x Anti-Target Antibody | 60 µL |
| Anti-GAPDH Antibody | 60 µL |
| HRP-Conjugated Anti-Rabbit IgG Antibody | 12 mL |
| HRP-Conjugated Anti-Mouse IgG Antibody | 12 mL |
| SDS Solution | 12 mL |
| Stop Solution | 24 mL |
| Ready-to-Use Substrate | 12 mL |
| Crystal Violet Solution | 12 mL |
| Adhesive Plate Seals | 2 seals |
The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:
- Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
- Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
- 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
- Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
- Graph paper or computer software capable of generating or displaying logarithmic functions
- Absorbent papers or vacuum aspirator
- Test tubes or microfuge tubes capable of storing ≥1 ml
- Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
- Orbital shaker (optional)
- Deionized or sterile water
*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1. | Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells. |
| 2. | Incubate the cells for overnight at 37°C, 5% CO2. |
| 3. | Treat the cells as desired. |
| 4. | Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice. |
| 5. | Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells. |
| 6. | Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4°C for a week. |
| 7. | Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature. |
| 8. | Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time. |
| 9. | Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature. |
| 10. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 11. | Add 50 µL of 1x primary antibodies (Anti-GluR4 Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature. |
| 12. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 13. | Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature. |
| 14. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 15. | Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark. |
| 16. | Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader. |
(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)
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